Advances in Phenazines over the Past Decade: Review of Their Pharmacological Activities, Mechanisms of Action, Biosynthetic Pathways and Synthetic Strategies

Phenazines are a large group of nitrogen-containing heterocycles, providing diverse chemical structures and various biological activities. Natural phenazines are mainly isolated from marine and terrestrial microorganisms. So far, more than 100 different natural compounds and over 6000 synthetic derivatives have been found and investigated. Many phenazines show great pharmacological activity in various fields, such as antimicrobial, antiparasitic, neuroprotective, insecticidal, anti-inflammatory and anticancer activity. Researchers continued to investigate these compounds and hope to develop them as medicines. Cimmino et al. published a significant review about anticancer activity of phenazines, containing articles from 2000 to 2011. Here, we mainly summarize articles from 2012 to 2021. According to sources of compounds, phenazines were categorized into natural phenazines and synthetic phenazine derivatives in this review. Their pharmacological activities, mechanisms of action, biosynthetic pathways and synthetic strategies were summarized. These may provide guidance for the investigation on phenazines in the future.


Introduction
Natural products are considered to be especially valuable resources for drug discovery. With the rapid development of technologies for isolation, purification and detection, great interest has been shown in the underexplored natural products. Natural phenazines are mainly discovered from microorganisms of marine and terrestrial. More than 100 natural phenazine derivatives and over 6000 synthetic phenazine derivatives have been investigated so far [1][2][3][4]. Phenazine derivatives are a large group of planar nitrogencontaining heterocyclic compounds and the most important core structure is a pyrazine ring (1,4-diazabenzene) with two annulated benzenes [5][6][7]. Phenazine derivatives differ in their chemical and physical properties based on the type and position of present functional groups. Their oxidation-reduction (redox) and fluorescent properties have attracted increasing attention. Some of them are significant dyes applied in medical and biological industry, while others are developed as efficient fluorescent probes to study the change of biochemical profile in vivo [8,9]. Natural phenazines are produced directly from various microorganisms, including Pseudomonas spp., Streptomyces spp., Actinomycete spp. in terrestrial and marine environments. They, like most other important secondary metabolites, possessed various biological activities and have been extensively studied for a long period of time [10,11]. Phenazines and their derivatives exhibit a broad range of biological activities, such as antimicrobial, antiparasitic, neuroprotective, insecticidal, anti-inflammatory, anticancer activity and so on [12][13][14][15]. Phenazine derivatives could be used as prodrugs due to biological activities, for which pharmacologists and chemists have committed themselves to make them into patent medicines. For example, clofazimine ( Figure 1) is successfully applied in clinic as widely used antileprosy and antitubercular drug due to antimicrobial activity and immunosuppressive properties [16]. XR11576, XR5944, NC-182 and NC-190 ( Figure 1) belong to fused aryl phenazine derivatives, also they show significant anticancer activity and are under clinical studies [7]. Phenazine derivatives display antibacterial activity mainly against methicillin-resistant due to redox properties [17]. According to reports in recent years, phenazine derivatives possessed antiproliferative activities against various cancer cell lines [18][19][20][21]. Additionally, phenazine derivatives were candidates to be developed as inhibitors of disease-related targets and reported to show activity of inhibition to multiple enzymes [22][23][24][25]. Although phenazine derivatives possessed a broad activity spectrum, the in-depth study was hindered due to the limited resource. Many research groups devoted themselves to carrying out the synthetic work to investigate biological activities of synthetic phenazine derivatives. Laursen et al. reviewed natural and synthetic phenazine derivatives with regard to biological activities in 2004 [6]. Phenazines and their derivatives had been associated with anticancer activity since 1959. On the basis of significant anticancer activity of phenazines and their derivatives, Cimmino et al. excellently reviewed natural and synthetic phenazines and derivatives about their anticancer activity and mechanisms of action in 2012, covering articles from 2000 to 2011 [26]. In recent years, researchers found various novel structures of natural phenazine derivatives and investigations of pharmacological activity were involved in many aspects. In this review, phenazines isolated from microorganisms, synthetic phenazine derivatives, their pharmacological activities and mechanisms of action were summarized, covering the articles from 2012 to 2021.

Natural Phenazine Derivatives
In the past decades, according to the published articles, many researchers investigated known phenazines deeply and further evaluated their potent biological activity. Other researchers tried to find novel phenazines from natural sources. Natural phenazines can be categorized according to the types of functional groups and their linking positions on the phenazine core.

Biological Activity of Known Phenazines
Compound 1 (phenazine-1-carboxylic acid, Figure 2) is also called tubermycin B due to its antibiotic activity against Mycobacterium tuberculosis. It is widely distributed in various microorganisms as a precursor of many natural phenazine derivatives. Gorantla et al. firstly reported its antifungal activity against major human pathogen, Trichophyton rubrum, which could be responsible for causing athlete's foot, jock itch, ringworm and fingernail fungus infections. The minimum inhibitory concentration (MIC) was 4 mg/mL [27]. Varsha et al. first isolated it from Lactococcus BSN307 and investigated its anticancer activity against HeLa cell line (IC 50 = 20 µg/mL) and MCF-7 cells (IC 50 = 24 µg/mL). It showed inhibitory activity towards leucine and proline aminopeptidases; thus, it would be used as a potential metalloenzyme inhibitor [28]. Compound 2 (Figure 2) is also a significant phenazine-type metabolite produced by various microorganisms. Cardozo et al. investigated its antibacterial activity against MRSA (Methicillin-resistant Staphylococcus aureus) strains and found its synergic effect when combined with silver nanoparticles produced by Fusarium oxysporum [29]. Thanabalasingam et al. first isolated it from the leaves of a medicinal plant Coccinia grandis [30]. Tupe et al. tested its activity against human pathogen Candida albicans (MIC = 32-64 µg/mL), demonstrating its mechanism of antibacterial and antifungal activities via reactive oxygen species (ROS)-mediated apoptotic death; 2 could lead to production of intracellular ROS. ROS caused hyperpolarization of mitochondrial membrane, following externalizing phosphatidylserine, chromatin condensation and DNA fragmentation, thus, inducing apoptosis and, finally, cell death [31]. Kennedy et al. and Ali et al. further investigated anticancer mechanism of 2. The anticancer activity mechanism was also connected with ROS. p53, Bax and cytochrome C (Cyto-C) were overexpressed while caspase-3 was activated and oncogenic, anti-apoptotic proteins such as poly ADP-ribose polymerase (PARP) and B-cell lymphoma-2 (Bcl-2) family proteins (Bcl-2, Bcl-w and Bcl-xL) were inhibited ( Figure 3) [32,33]. Pyocyanin (7, Figure 2) is a redox-active phenazine. It is a major virulent factor produced by Pseudomonas aeruginosa, which exerts damage effects on mammalian cells. Chai et al. explored pathogenesis of 7 on macrophages. Biological data showed it could promote IL-8 secretion and mRNA expression in a concentration-dependent manner. Signal pathways of the protein kinase C (PKC) and nuclear factor-κ-gene binding (NF-κB) were involved in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells infected by 7 [34]. Forbes et al. aimed to investigate the pyocyanin role of redox-sensitive mitogenactivated protein kinase (MAPK) by inducing toxicity in A549 cell line. The results showed that pyocyanin-induced cytotoxicity was different from c-Jun N-terminal Kinase (JNK) and p38MAPK signaling pathways. Acute ROS production and subsequent oxidative stress strengthened its toxicity [35]. 1,6-Dihydroxyphenazine 5,10-dioxide (8, Figure 2) is also called iodinin. It was discovered to show anti-bacterial activity and weak activity against a mouse tumor model. Sletta et al. firstly isolated it from Streptosporangium sp. DSM 45942 from the fjord sediment; 8 showed great antibacterial and antifungal activities against Candida glabrata and Enterococcus faecium, MIC ranging from 0.35-0.71 µg/mL. Compared with normal rat kidney (NRK) fibroblasts, 8 showed higher selectivity towards leukemia cell line. It was a promising compound to be developed as an anticancer drug, especially those targeting leukemia [36]. Myhren et al. further investigated its anticancer potential against acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. The results demonstrated its anticancer potency against two selective cancer cell lines and weak toxicity to normal cells. Molecular modeling results suggested that it could intercalate between bases in the DNA, leading to DNA-strand break. The apoptosis progress was associated with Fms-like tyrosine kinase (FLT3) internal tandem duplications, mutated/deficient p53 and activation of caspase-3 [37]. Hifnawy et al. isolated compounds 1, 5 (phenazine-1,6-dicarboxylate) and 6 (phencomycin) from actinomycetes, Micromonospora sp. UR56 and Actinokineospora sp. EG49. These compounds demonstrated high to moderate antibacterial and antibiofilm activities against four bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa), with modest cytotoxicity against four cell lines (WI38, HCT116, HepG-2 and MCF-7). They took Staphylococcus DNA gyrase-B and pyruvate kinase as targets. Subsequently, in vitro data showed that 1, 5 and 6 ( Figure 2) exerted their bacterial inhibitory activities through inhibiting Staphylococcus DNA gyrase-B and pyruvate kinase [39].

Terpenoid Phenazines
Terpenoid phenazines contain common structural feature of isoprenylated C or N side chains and most of them show moderate or weak antibacterial activity. Kondratyuk et al. isolated marine phenazines 9 and 10 ( Figure 4) from Streptomyces sp. strain CNS284: 9 demonstrated inhibitory activity of NF-κB and cyclooxygenase-2 (COX-2); 10 showed potent (sub-µM) inhibition activity of prostaglandin E2 (PGE2) production. However, these activities monitored did not have a strong correlation with each other. The mechanism of action was not apparent and needed to be further investigated [40]. Ohlendorf et al. isolated geranylphenazinediol (11, Figure 4) from a marine sediment Streptomyces sp. strain LB173 [41]: 11 bears geranylation at C-4 side. It showed weak antibacterial activity and great inhibitory activity toward human acetylcholinesterase (IC 50 = 2.62 ± 0.35 µM). Phenaziterpenes A (12) and B (13, Figure 4) are structurally related to geranylphenazinediol, bearing O-geranylation. Song et al. isolated them from Streptomyces lusitanus SCSIO LR32. However, compounds 12 and 13 did not show antibacterial activity and cytotoxicity against tumor cell lines [42].

Glycosylated Phenazines
A few natural glycosylated phenazines have so far been found and reported. The activity of glycosylated phenazines was not remarkable and needed to be further investigated. biological assay data demonstrated that the weak cytotoxic activity was not associated with DNA intercalations and topoisomerase inhibition. The mechanisms of action were uncertain and needed to be further investigated [45]. subtilis. In addition, 26-28 and 30 also showed antimicrobial activity against Gram-negative E. coli. Interestingly, they found that glycosylated 27 and 28, compared with their corresponding aglycone, displayed enhanced activities against Gram-negative E. coli [43].

Divergent Fused Phenazines
This class of phenazines contains more than one phenazine-derived moiety. There are a few divergent fused phenazines in nature that have been reported so far. Li et al. isolated diastaphenazine (31, Figure 6) from an endophytic Streptomyces diastaticus subsp. ardesiacus: 31 was a cytotoxic dimeric phenazine, showing antibacterial activity against S. aureus (MIC = 64 µg/mL). However, 31 was inactive against E. coli and C. albicans even at 128 µg/mL. Compared with positive control (adriamycin), 31 showed weak cytotoxicity against HCT116, BGC-823, HepG2, HeLa and H460 cell lines with IC 50 values of 14.9 µM, 28.8 µM, 65.2 µM and 82.5 µM, respectively [46]. Baraphenazines A−C (32-34, Figure 6) are fused 5-hydroxyquinoxaline/alpha-keto acid amino acid compounds. Baraphenazines D and E (35 and 36, Figure 6) are special diastaphenazine-type compounds. In addition, baraphenazines F and G (37 and 38, Figure 6) are phenazinolin-type compounds. Wang et al. isolated them from Streptomyces sp. PU-10A and investigated their anticancer activity. Only 36 displayed appreciable activity against A549 and PC3 cell lines with IC 50 values of 2.4 µM and 4.7 µM, respectively. Structure-activity relationship (SAR) indicated that the group of amide on 36 was important to the anticancer activity. On the contrary, the group of free acid on 32−35, 37 and 38 was not benefit to the antiproliferative activity. These bioactivity data could explain a general toxicity-based mechanism of action [47].

Biological Activity of New Phenazines
Kennedy et al. isolated 5-methyl phenazine-1-carboxylic acid (39, Figure 7) from a rhizosphere soil bacterium. It showed selective cytotoxicity against A549 and MDA MB-231 cell lines in a dose-dependent manner, with IC 50 values of 488.7 nM and 458.6 nM, respectively. It exhibited antiproliferative activity by inhibiting cell viability, DNA synthesis and induced G1 cell cycle arrest and apoptosis in cancer cell lines. It was mediated by mitochondrial apoptotic pathway via activation of caspase-3 and down regulation of Bcl-2 expression [32].

The Progresses of Biosynthetic Pathways of Phenazines
McDonald et al. found that 2-amino-2-deoxyisochorismic acid could be completely converted into 1. These compounds were mainly extracted from Pseudomonas spp. PhzB, phzD, phzE, phzF, phzG and so on, which belong to the phz gene family and they were proved to play important roles in phenazine synthesis [49]. Chorismic acid (56) was not only a common precursor for many primary and secondary metabolism but also the first substrate in biosynthetic pathway towards natural phenazines. Many important phenazines could be produced from microorganisms by this biosynthetic pathway. Combining with previous reports of Xu et al. and Blankenfeldt et al., the classical biosynthetic pathway towards strain-specific phenazines starting from chorismic acid is shown in Figure 8 [5,50].
Normally, the biosynthetic pathway in Pseudomonas mainly focused on simple modification of phenazine cores. Shi et al. reported a different biosynthetic pathway of various complex phenazines from the entomopathogenic bacterium Xenorhabdus szentirmaii. By modifying the core structure of phenazine, such as electron-rich aromatic rings, reduced form nitrogen(s) and carboxylic acid, a variety of natural phenazine derivatives can be generated. The synthesis of compound 59 is controlled by the typical phz operon in X. szentirmaii similar to classical biosynthetic pathway. Further modification of 59 was diversified by the enzymes from two discrete biosynthetic gene clusters. This progress of biosynthetic pathway was involved in multiple enzymatic and non-enzymatic reactions ( Figure 9) [51].
Guo et al. developed a biosynthetic pathway to synthesize phenazine N-oxides in Pseudomonas chlororaphis HT66 ( Figure 10). They used three enzymes, a monooxygenase (phzS), a monooxygenase (phzO) and the N-monooxygenase (naphzNO1). Additionally, naphzNO1 only catalyzed the conversion of 80, but failed to convert into 81 in vitro. This study also provided a promising method for the synthesis of aromatic N-oxides by naphzNO1 [52].

Synthetic Phenazine Derivatives
Although natural phenazines possess a variety of biological activities, most of which show moderate or weak activity, thus lacking the possibility to be used as drugs. Structural modification and total synthesis are used to achieve some phenazine derivatives which show notable activity. Normally, the researchers focus on enhancing one special biological activity. Here, synthetic phenazine derivatives will be classified into the following categories in detail, with the perspective of biological activities and functional groups connected to phenazine core.

Halogenated Phenazine Derivatives
According to related reports, bacterium would stop growth in MIC of 2-4 µg/mL and die in MIC of 2 µg/mL [48]. Halogenated phenazines derivatives are tested as antibacterial agents which could target multiple persistent bacterial phenotypes effectively and show negligible toxicity against mammalian cells. Antibacterial effect of halogenated phenazine derivatives could be attributed to membrane disruption, interference with redox cascades or electron-flow and the production of ROS [13,53]. Halogenated phenazine derivatives needed to be further developed by chemists due to the great antibacterial activity.
Conda-Sheridan et al. synthesized a series of phenazines derivatives inspired by some natural halogenated phenazines. They found N-(methylsulfonyl) amide group in the position of C-4 and halogenated group in the position of C-6 would remarkably improve the activity against MRSA. Compounds 82 and 83 ( Figure 11) showed stronger antibacterial activity in these synthetic halogenated phenazines compared to positive drug vancomycin (MIC = 2 µg/mL). The mechanism of action of the most active compound was also investigated, but various tested biological data indicated that 83 did not have correlations with major reported antibacterial mechanisms. The in vitro IC 50 (83), respectively; 83 seemed to be a promising molecule for the development of MRSA drugs. Additionally, the application of computational methods such as quantitative structure-activity relationship (QSAR) and the prediction of LogP would promote the development of antibacterial drugs [54].   Figure 13); 87 was selected as a lead antibiotic which displayed great inhibitory activity against S. aureus (MIC= 1.56 µM). For example, the activity of 88 increased two folds by systematic structural diversification and the SAR was discussed as shown in Figure 13 [56].

Derivatives of Clofazimine
Although clofazimine (Figure 14) is an antibiotic against multidrug-resistant M. tuberculosis, the clinical utility of this agent is limited by its poor physical and chemical properties and the possibility of skin discoloration. TBI-1004 and B4100, modified at different positions (Figure 14), showed stronger anti-M. tuberculosis activity than clofazimine. Zhang et al. designed and synthesized a series of riminophenazine derivatives which contained a pyridyl group at the C-3 position of the phenazine core, inspired by previous investigations about the developments of TBI-1004 and B4100. Among these derivatives, compound 97 ( Figure 14) demonstrated similar activity against M. tuberculosis. Additionally, reduced the possibility of skin discoloration in an experimental mouse infection model as compared to clofazimine. In addition, physicochemical properties and pharmacokinetic profiles of 97 were improved [57].

Water-Soluble Triazole Phenazine Derivatives
Hayden et al. evaluated water-soluble triazole phenazine derivatives, which were synthesized previously. Compounds 141-143 ( Figure 18) showed high antimicrobial activity at tested concentrations without cytotoxicity against human epithelial cells and tested biological data suggested that 141-143 could interrupt metabolic electron-transfer cascades thereby exhibiting cytotoxicity against E. coli, rather than production of ROS [64].

Insecticidal Activity
Podophyllotoxin is a natural product used as the lead compound for the preparation of insecticidal agents. It contains A, B, C, D and E rings. Zhi et al. designed and synthesized a series of podophyllotoxin-based phenazine derivatives modified in the C, D and E rings. In addition, the insecticidal activity of target compounds was investigated which showed insecticidal activity against Mythimna separata Walker in vivo. Compounds 148 and 149 (Scheme 3) were phenazine derivatives of 4-acyloxypodophyllotoxin modified in the E ring. They demonstrated stronger insecticidal activity than toosendanin [65]. Then, they designed and synthesized a series of oxime derivatives of podophyllotoxin-based phenazines modified in the C, D and E rings. Compounds 153-157 (Scheme 4) exhibited equal or higher insecticidal activity than toosendanin. The combination of podophyllotoxin and phenazine was proved to enhance insecticidal activity [66].

Antiparasitic Activity
Chagas' disease, caused by Trypanosoma cruzi, is a widely spread endemic disease in American. Alvarez and Minini et al. selected phenazine 158 (Figure 19) from their own chemistry library and investigated its in-depth insight mechanism of inhibition; 158 could bind to a widespread enzyme, triosephosphate isomerase (TIM) from T. cruzi. It showed great inhibitory activity against TIM and could be further developed as inhibitors of TIM; 158 showed highly selective inhibition against T. cruzi enzyme (TcTIM) and weak inhibition against T. brucei (TbTIM), without affecting TIM from H. sapiens (HsTIM) and Leishmania sp. (LmTIM) [67,68].  Table 1. Hernández et al. investigated chemosensitizer effect of compounds 159 and 161 to cisplatin. They showed a significant increase of the antiproliferative activity compared with the control group treated with cisplatin alone, demonstrating sensitization to cisplatin therapy. In addition, 159 combined with cisplatin induced cell cycle arrest on bladder cancer cells, resensitizing the invasive and cisplatin resistant 253 J cell line. It also showed great inhibition activity against histone deacetylase (HDAC) and sensitized chemotherapeutic drugs to better access to DNA, which would cause DNA damage, leading to cell death [69]. Phenazine 5,10-dioxide derivatives have also been reported in development as bioreductive agents. This class of compounds all contain a bioreductive moiety, the N-oxide group and a planar heterocycle moiety, phenazine [70,71]. After the hypoxic selective bioreductive process, the phenazine moiety can interact with DNA causing cytotoxicity in the solid tumour cells [72]. Gonda et al. attempted to find selective hypoxic cytotoxins with additional ability to inhibit DNA topoisomerase II. Inhibitive values in normoxia and hypoxia condition of these compounds were shown in Table 1: 162-164 displayed some degree of selectivity; 165 showed non-selectivity towards both conditions, normoxia and hypoxia. Meanwhile, 159 and 163 showed the best selectivity; 164 exhibited inhibition of topoisomerase II in hypoxia; 166 showed no inhibition of topoisomerase II in hypoxia and normoxia. The DNA interaction abilities of phenazine 5,10-dioxide derivatives were related to cytotoxicity in normoxia or hypoxia. SAR implicated that the arylethenyl moieties were generally responsible for normoxic cytotoxicity. On the contrary, the group of sulfonamido did not produce selective cytotoxicity whether in normoxia or hypoxia [73].

Benzo[a]phenazine Derivatives
According to related reports, benzo[a]phenazine derivatives show significant activity of antiproliferation against HL-60 cell line. Topoisomerases, including topoisomerase I and II, have been proved to be effective anticancer targets in drug discovery due to highly over-expression in cancer cells [74,75].
Benzo[a]phenazine derivatives 167-174 are listed in Table 2 Compared with 167, 168 and 169 were introduced a methoxy group, their inhibitory activity was significantly improved and there was a good correlation between the inhibitory activity and cytotoxic activity. Caspase-3/7 activation assay showed that this class of compounds could induce an apoptotic response in HL-60 cell line [76].  The relative Topo I cleavage complex stabilizing potencies of the compounds are presented as follows: −, no detectable activity; +, weak activity; ++, weaker activity than that of CPT; +++, activity similar to that of CPT.; c The relative Topo II ATPase inhibitory potencies of the compounds are presented as follows: −, no detectable activity; +, weak activity; ++, weaker activity than that of 1,4-naphthoquinone; +++, activity similar to that of 1,4-naphthoquinone, higher activity than that of 1,4-naphthoquinone; ++++.; Nd = not determined.
Yao et al. synthesized a series of 7-alkylamino substituted benzo[a]phenazine derivatives. Most of these compounds showed better inhibitory activity in HL-60 cell line than the other tested cell lines. The structure-activity relationship studies revealed that the substitution of amino groups on terminal of side chain at N-7 position could improve the Topo I/II inhibitory activity and cytotoxicity: 170-172 with the dimethylamino terminal showed good Topo I and Topo II inhibitory activity; 170 could stabilize the Topo I-DNA cleavage complexes in vivo; 173 with methoxy group at position C-9 exhibited good Topo I and Topo II inhibitory activity at 25 mM concentration; 173 showed inhibition of ATPase [22].

Pyran[2,3-c]phenazine Derivatives
Phenazine derivatives and pyran derivatives are important heterocyclic compounds which possess good biological activity. The heterocyclic pyran structure usually is a functional framework which appears in amounts of important drugs and natural products. Molecular hybridization strategy shows great prospect in the present drug discovery to reduce the side effects and the occurrence of drug-resistance [77]. The aromatic interlayer coupling structure of phenazine, as well as the structural characteristics of fluted or specific enzyme binders, leads to selective high-affinity binders that target DNA and DNAenzyme complexes [78]. According to recent articles, pyran [2,3-c] phenazine derivatives mainly showed cytotoxicity against HepG2 cell line. In addition, the mechanism inducing apoptosis against cancer cells about this class of compounds is shown in Figure 3. In addition, they also used experimental mouse models to test in vivo activity of these phenazine derivatives. Among these phenazine derivatives, 181 was selected to do tumor xenografts experiment to test the effect of inhibition. H22 cells was injected into ICR mice, inhibitions were 7.78% (5 mg/kg), 68.89% (10 mg/kg) and 77.78% (20 mg/kg), respectively. Further mechanism studies implicated 181 acted as topoisomerases I and II dual inhibitor, cell cycle arrester and apoptosis inducer against HepG2 cell line [23].

Benzo[a]pyran[2,3-c]phenazine Derivatives
This class of phenazine derivatives, which contain phenazine, pyran and benzo core, possess great anticancer activity but rarely reported. Inspired by XR11576 (Figure 1), benzophenazine derivative has been proved to be a great antitumor compound which could be further modified. Gao

Benzo[a]chromeno[2,3-c] phenazine Derivatives
Chromenes are also an important class of heterocyclic compounds which exhibit attractive pharmacological properties, such as antitumor, anti-vascular, antioxidant, antimicrobial, sex pheromone, tumor necrosis factor-α (TNF-α) inhibitor, cancer therapy and central neuroprotective activities [80]. Chromenes and phenazine derivatives both have attracted attention, but benzo[a]chromeno[2,3-c] phenazine derivatives have rarely been investigated [81]. Reddy   Notably, these active compounds showed no cytotoxicity on the epithelial cells from the 293T non-cancer cells. Moreover, mechanism of 195 was similar to control drug (cisplatin), inhibiting cancer proliferation by inducing apoptosis [82]. According to the report of Gao et al., Kale et al. selected some 2-phenazinamines derivatives and further utilized computational methods to investigate their protein targets. The experimental data of 199-202 showed great binding energy against BCR-ABL tyrosine kinase by Autodock 4.2. Scores of 199-202 were −7.6 kcal/mol, −8.8 kcal/mol, −7.2 kcal/mol and −7.1 kcal/mol, respectively. In addition, the score of imatinib was −8.7 kcal/mol. All the computational data showed 2-phenazinamine derivatives would be inhibitors against BCR-ABL tyrosine kinase and needed to be further investigated [25].

Derivatives Derived from 2,3-Diaminophenazine
Protein kinases (PKs) are essential in many cellular processes, which catalyze phosphorylation of different cellular substrates. Then, phosphorylation in turn regulates various cellular functions. Normally, their activity is strictly regulated. Under pathological conditions, PKs can be deregulated, leading to changes in the phosphorylation, resulting in uncontrolled cell division, inhibiting apoptosis and other abnormalities. Various cancers are known to be caused or accompanied by deregulation of the phosphorylation. Screening new potent, selective and less toxic compounds to inhibit PKs has been proved to be a promising cancer treatment strategy [83].

2,3-Dialkoxyphenazine Derivatives
Endo et al. reported the only antitumor activity of 2,3-disubstituted phenazines against sarcoma and carcinoma tumors in 1965 [84]. The presence of long fatty chains could provide a good effect to cross the lipid barrier [85]. Moris et al. synthesized 2,3-dialkoxyphenazine derivatives using an easy, efficient and straightforward condensation method. These compounds 208-211 ( Figure 24) were firstly reported to show activity on MiaPaca pancreatic cell lines with IC 50 of 0.06 µM, 21 µM, 75 µM and 7 µM, respectively. Interestingly, 208 and 209 interacted with DNA through hydrogen bonds remarkably, showing significant anticancer activity. Compared to Gemzar ® , 208 and 211 were the most effective ones against pancreatic MiaPaca cell resistant lines. The experimental results showed that the carboxyl substituents on position 7 did not interact with each other through hydrogen bonds. Although possessing planar structures, these derivatives did not have similar mechanism of action as Gemcitabine. In vivo study on mice, 211 was as efficient as Gemzar ® at a ten times lower concentration (1 mg/kg vs. 10 mg/kg) [21].

Conclusions
In this review, we introduced natural phenazines and synthetic phenazine derivatives, which were reported from 2012 to 2021. The biosynthetic pathways of natural phenazines, sources of microorganism and operon genes were illustrated in detail. Additionally, their pharmacological activities, mechanisms of action and structure-activity relationships were also summarized. In future studies, first of all, it is still necessary to find novel structures from natural sources for the screening of lead compounds. Secondly, it is very important to design and synthesize new compounds based on existing SAR through structural modification. The successfully applied drug clofazimine and XR11576, XR5944, NC-182 and NC-190 in clinical studies are good references. The structure modification at C-2, C-3, C-4 and N-6 sites and ring fused derivatives have a good prospect. In addition, target-based drug design can reduce the randomness. Finally, more extensive activity screening will enable more efficient use of compound resources.
Author Contributions: J.Y., investigation, visualization, writing-original draft, writing-review and editing; W.L., investigation, visualization, writing-review and editing; J.C., investigation, visualization, writing-original draft, writing-review and editing; Y.W., investigation, visualization, writing-original draft, writing-review and editing; D.L., funding acquisition, project administration, supervision, writing-original draft, writing-review and editing; H.H., project administration, supervision, writing-original draft, writing-review and editing; H.C., funding acquisition, project administration, supervision, writing-original draft, writing-review and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This paper was financially supported by the Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.

Conflicts of Interest:
The authors declare no conflict of interest.